1

Supplementary materials

Table S1 Primer sequences used in this study

Primers Sequence (5’ to 3’) GenBank Accession No.

MYB-F AARAGYTGYAGATTRAGGTGG

MYB-R CCARTAGTTTTTSACATCGTT

SmMYB9b-F TCCCTCCTTTATCCTCTTCCAC

SmMYB9b-R TAGGGTTTATGTATCCTTTGTGA

SmMYB9b-SpeⅠ-F GCTACTAGTGAGCGAAATTAACACGAGATGG

SmMYB9b-BstEⅡ-R ATAGGTGACCAAGAAGTCAAGCATTGGTGT

Actin-F GAACTGGAATGGTCAAGGCTGGATT HM051058

Actin-R AGTTGCTGACAATACCGTGCTCAA

AACT-F GGAGAGGACGACCATCCACCATTGT EF635969

AACT-R GGCATGAGCGGCATCAGCATATCC

CMK-F TACACCAACGCCACTCTTCCTCAA EF534309

CMK-R GCCGTCGCTCTGATAAGATGGATTC

CPS1-F TGTTGGTGAAGTTGGTGCTTGAGAA EU003997

CPS1-R CTATGGTGTCGGCGGCATGATAC

CPS5-F TAGAAGATGCAGCTACTTTCTCTGCT KC814642

(Cui et al., 2015) CPS5-R CATCATCTTCACCGCCGTACTGTT

DXR-F GAGGAGATCATCCACTACGACCAGT DQ991431

DXR-R CACATCCATCAACCTCCGCTCATAC

DXS1-F GGAAGAAGAGACAGGATGCCGAGTT EU670744

DXS1-R AGTCCAGGTAGCCAGCATTGTTCAT

DXS2-F GATGGCGTCGTCTTGTGGAGTTAT FJ643618

DXS2-R CGTCGTTGGTGTTATCCTGTTGAAG

FPPS-F GTGGACTGATGGTTCTCGTCAATGG EF635968

FPPS-R CACACCAGCCAAGAGCACTAGC

GGPS-F CGTCGTCGAGAAGGCGAATCAC FJ643617

GGPS-R GGCGGAGGTCATCATTGTCCATAC

HMGR1-F GCTCTGCTATTGCTGGTGCTCTTG GU367911

HMGR1-R CCTCCGACAGTGCCAACCTCAA

HMGR2-F CGTCGCCTCCTTCATCTATCTCCT FJ747636

HMGR2-R TGATCTCGTCGTCGTCGTCCAA

HMGR3-F CTCACATTCGTCGCCTCCTTCATCTA JN831102

HMGR3-R TTCCTCGTCTTGCTCGTCGGTAAC

HMGS-F CGACCTTGCCAGTGAATATCCAGTT FJ785326

HMGS-R TGCTTGCCCTCCTGCTTCTCA

HDR-F TGCCGTCGATGAGATGAGGATT JN831100

HDR-R AAGCAGTAGCCACAGTCTCTTCA

HDS-F TCGTGATGGTTCGGTTCTGATGTCA JN831098

HDS-R GAGTCCACTGTTGCGAGGTCCTT

2

IPPI-F AATGTCGTCCTTGACCAGCATCC EF635967

IPPI-R GAGGTAGCGGCGGTGAATGAAG

KSL1-F AAGAGAAGCCAGCCGCAGAATATG EF635966

KSL1-R CATCCAAGGTTAGTGCCGTGTCAT

KSL2-F TTAGTTTTGGAGGGCAAGAAGAGTGT KC814643

(Cui et al., 2015) KSL2-R CTCCTGTTTGGTCGTTGAGAAGAATA

MCT-F AGGTTCTGAAGGATGGCAAGCGAAT JN831096

MCT-R CCATAGCGTCTTCCTGTCCAGTGTT

MDS-F GAGCACGGTTGGAGCAGAGACT JN831097

MDS-R TGAGGAATATTGATGCCGCCGATAATG

MK-F CTGCGTTCTCACACTGCTACCTACT JN831104

MK-R GCTAATCTCCATGCCTCTTCCACCTAT

MDC-F AAGCACTGGGATGACCTCGTTAT JN831105

MDC-R ATGGAACTGATTACTATCGGCACAAG

PMK-F GCTTGCCATTGACCTCGGAATCT JN831095

PMK-R ACCACTGAACTCTGAAGGAAGACTGA

3

Table S2 SmMYB9b transcripts, biomass and secondary metabolites in individual SmMYB9b-OX and control hairy roots of S. miltiorrhiza

Items SmMYB9b-OX Control

M1 M11 M5 M4 M13 C3 C4 C6 C7 C8 C13 C15 C16

SmMYB9b transcripts 59.50±9.98 36.94±3.10 28.25±1.66 26.38±11.35 22.74±6.14 1.00±0.14

Biomass (DW, g) 0.29±0.02 0.29±0.01 0.33±0.05 0.35±0.02 0.42±0.04 1.11 1.02 0.82

Tanshinones

(mg/g DW)

CT 0.36 0.24±0.04 0.29±0.03a 0.31±0.07 0.27±0.05 0.03 0.04 0.05 0.01 0.05 0.04 0.03 0.02

TA-I 1.89 1.05±0.33 1.55±0.17a 1.51±0.41 1.33±0.13 0.47 0.49 0.48 0.41 0.56 0.53 0.46 0.43

TA-IIA 0.58 0.30±0.09 0.40±0.02a 0.43±0.10 0.42±0.03 0.08 0.58 0.59 0.28 0.68 0.66 0.53 0.42

TTA 2.83 1.58±0.44 2.23±0.19a 2.25±0.58 2.02±0.13 0.57 1.11 1.12 0.70 1.28 1.23 1.01 0.88

Anthocyanins

(QAnthocyanins = (A530 - 0.25 ×

A657) × M−1

) (Absorbance

units (AU)/g DW)

4.74±0.50 3.39±0.12 3.99±0.14 4.28±0.15 5.57±0.07 3.27±0.04

Lignins (mg/g DW) 14.74±0.12 13.40±0.14 13.90±0.12 14.30±0.14 15.61±0.04 13.27±0.50

Salvianolic acids

(mg/g DW)

RA 74.83±12.63 63.50±4.38 58.91±5.60 38.12±3.02 51.62±0.20 105.41±0.36

SAB 86.91±4.84 90.72±1.6 101.36±7.38 32.95±4.12 79.35±1.73 89.48±5.87

TSA 161.74±17.46 154.22±5.99 160.27±12.98 71.07±7.13 130.97±1.93 194.89±6.23

Note: a These data were from two independent cultured samples. All the other data with SD were from three independent cultured samples of the same hairy root

line. Data without SD were from one cultured samples. M1, M11, M5, M4, and M3 stand for individual SmMYB9b over expressing (Gene-Modified) hairy root lines.

C3, C4, C6, C7, C8, C13,C15, and C16 stand for individual Control hairy root lines.

4

Table S3. Characterization of diterpenoids from SmMYB9b-OX danshen hairy roots identified

with LC-MS

Compound

No.

tR

(min) Assigned identity

Molecular ions

m/z (Da) Fragment ions m/z (Da)

1 19.24 unknown 487.50 469.97, 425.10, 371.07

2 25.66 15,16-dihydrotanshinone I 278.90 260.73, 232.72

3 27.29 1,2-didehydromiltirone 280.91 262.64, 234.78, 220.76

4 28.32 methyltanshinonate 338.90 278.56, 260.67

5 31.59 cryptotanshinone 296.90 278.70, 263.73, 250.80,

235.75, 222.78, 208.76

6 32.48 tanshinone I 276.90 248.76, 220.74, 192.78

7 34.82 1,2-dihydrotanshinone I 278.90 260.70, 232.73

8 38.71 tanshinone IIA 295.10 276.71, 248.81, 233.74,

220.74

Note: “tR” is the abbreviation of “Retention time in HPLC assay”.

MS analysis was performed with a LCQ ion trap instrument (Thermo Finnigan, San Jose, USA)

equipped with an Xcalibur workstation using the reported program (Hu et al., 2005; Liu et al.,

2007). The positive ion mode for MS analyses was selected, working under the following

conditions: capillary voltage 19 V, spray voltage 5.0 kV, capillary temperature 300oC, sheath

gas flow rate at 40 (arbitrary units), auxiliary gas flow rate at 20 (arbitrary units), and tube

lens offset 40 V. The full scan mass spectra were recorded in the range of m/z 150-800. The

isolation width of precursor ions was 1.0 Th. The HPLC/MS data were acquired and

processed using the Finnigan Xcalibur 1.3 software provided by the manufacturer.

5

Table S4 Tanshinone concentration in one year danshen plants

Plant tissue/organs Concentration of tanshinones (μg/g DW)

CT TA-I TA-IIA

flower buds 1.2 1.4 1.5

blooming flowers 1.2 1.0 1.2

leaves 2.0 1.1 1.6

stems 1.1 0.9 1.2

roots 801.7 231.2 723.0

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Table S5 Phenylpropanoid concentration in danshen hairy roots

Items SmMYB9b-OX Control

Anthocyanins

(QAnthocyanins = (A530 - 0.25 × A657) × M−1)

4.48±0.08 (1.3) 3.27±0.04

Lignins (mg/g DW) 14.39±0.11 (1.1) 13.27±0.04

Salvianolic acids

(mg/g DW)

RA 57.39±5.16 (54%) 105.41±0.36

SAB 78.26±3.93 (87%) 89.48±5.87

TSA 135.65±9.10 (70%) 194.89±6.23

Note: Salvianolic acids and tanshinones of danshen hairy roots were isolated and measured

as reported (Zhao et al., 2011). Briefly, hairy roots were lyophilized and 50 mg of sample

powder was extracted in 5ml of 75% methanol with 0.1% (v/v) formic acid under sonication

for 40 min. Sample extracts were then centrifuged at 12,000g for 10 min and the supernatant

was filtered through 0.22µm membrane before analysis (Hu et al., 2005; Chen et al., 2006).

Reference standards of rosmarinic acid (RA) and salvianolic acid B (SAB) (Shanghai R&D

Center for Standardization of Chinese Medicines, China) were used to determine the

concentration of corresponding compounds. Total salvianolic acids were expressed as the

sum of SAB and RA. HPLC analysis was carried out with a Beckman CoulterTM ODS column

(250×4.6×5mm). A gradient elution of solvent A (acetonitrile) and solvent B

(water/acetonitrile/formic acid=90:10:0.4) at a flow rate of 1.0ml/min was used as follows:

0–40min, A 0%–30%, B 100%–70%. The injection volume was 10 µl and the detection

wavelength was 280nm.

Extraction and quantification of anthocyanins was performed according to reference

(Mehrtens et al., 2005) with minor modifications. Briefly, 1ml of acidic methanol (1% [w/v]

HCl) was added to 20 mg of dry hairy root powder and kept at 25℃ for 18 h under shaking

(120 rpm). Sample extracts were then centrifuged at 12,000 g for 5 min at room

temperature, 400µl of the supernatant was taken out and the volume was adjusted to 1ml

with 600µl of acidic methanol. Absorption of the extracts at wavelengths of 530 and 657 nm

was determined with spectrophotometer (HITACHI U-2900). Concentration of anthocyanins

was calculated using the following equation: Q=(A530-0.25*A657)/g of DW, where Q is the

concentration of anthocyanins, A530 and A657 are the absorptions at the wavelengths

indicated, and g is the dry weight of the hairy roots used for extraction.

Lignin was extracted and quantified according to reference (Xue et al., 2008) with minor

modifications. In short, 50mg of hairy root power was weighted and put in a tube. Then,

15ml of double distilled water was supplemented into the tube and kept at 65℃ water

7

bath for 30min with shaking from time to time. The suspension passing through with 0.45

microns nylon membrane was weighted and the residue was successively washed with

water, absolute ethanol, acetone and diethyl ether. The residue and membrane were then

dried in a vacuum oven for 48h under 40℃, treated with P2O5 overnight, and weighted.

One milliliter of 25%[V/V] acetyl bromide/glacial acetic acid was added to 5mg of

hydrolysate and kept at 70℃ for 30min. After cooling on ice, the solution was mixed with

5ml of glacial acetic acid and then centrifuged at 12,000 g for 5min. Thirty microliter of the

supernatant was mixed with 1.5 M NaOH, 0.5 M hydroxylamine hydrochloride and acetic

acid. The 280 nm absorbance of the mixture was measured. For lignin quantification the

following equation was used: X=(A280-0.0009)/23.077, where X is the lignin concentration

and A280 is the absorption of extract at 280nm.

Chen, J., Wang, F., Lee, F.S.C., Wang, X. and Xie, M. (2006) Separation and identification of

water-soluble salvianolic acids from Salvia miltiorrhiza Bunge by high-speed counter-current chromatography and ESI-MS analysis. Talanta, 69, 172–179.

Cui, G., Duan, L., Jin, B., et al. (2015) Functional Divergence of Diterpene Syntheses in the Medicinal Plant Salvia miltiorrhiza. Plant Physiol., 169, 1607–18.

Hu, P., Luo, G.-A., Zhao, Z.-Z. and Jiang, Z.-H. (2005) Quantitative determination of four diterpenoids in Radix Salviae Miltiorrhizae using LC-MS-MS. Chem. Pharm. Bull. (Tokyo)., 53, 705–9.

Mehrtens, F., Kranz, H., Bednarek, P. and Weisshaar, B. (2005) The Arabidopsis transcription factor MYB12 is a flavonol-specific regulator of phenylpropanoid biosynthesis. Plant Physiol., 138, 1083–1096.

Xue, F.C., Chandra, R., Berleth, T. and Beatson, R.P. (2008) Rapid, microscale, acetyl bromide-based method for high-throughput determination of lignin content in Arabidopsis thaliana. J. Agric. Food Chem., 56, 6825–6834.

Zhao, S., Zhang, J., Yang, L., Wang, Z. and Hu, Z. (2011) Determination and biosynthesis of multiple salvianolic acids in hairy roots of Salvia miltiorrhiza. Acta Pharm. Sin., 46, 1352–1356.

8

Table S6 Correlation analysis of SmMYB9b transcripts with phenylpropanoids from individual

SmMYB9b-OX and control hairy roots

Correlation

coefficiency

SmMYB9b

Transcripts Anthocyanins Lignin RA SAB TSA

SmMYB9b

Transcripts 1

Anthocyanins 0.331 1

Lignin 0.324 0.999** 1

RA -0.310 -0.504 -0.499 1

SAB -0.061 -0.220 -0.241 0.565 1

TSA -0.136 -0.405 -0.415 0.879* 0.890* 1

*. Correlation is significant at the 0.05 level (2-tailed). **. Correlation is significant at the 0.01 level (2-tailed).

9

Fig. S1 Schematic map of p35S::SmMYB9b.

p35S::SmMYB9b

10600 bp

Lac Z alpha

kanam ycin (R)

hygrom ycin (R)

Sm MYB9b

pVS1 sta

T-Border (r ight)

pBR322 bom

T-Border (le ft)

Nos poly-A

CaMV35S polyA

CaMV 35S prom oter

CaMV35S prom oter

pBR322 ori

pVS1 rep

BstEII (813)

SpeI (15)

10

Fig. S2 Genomic organization of SmMYB9b and Alignment of SmMYB9b with SmMYB9

A: Genomic organization of SmMYB9b; B: Alignment of the open reading frame (ORF)

regions of SmMYB9b and SmMYB9. C: Alignment of the amino acid sequences of SmMYB9b

and SmMYB9. Consensus nucleotides and amino acid residues are indicated in red letters.

1 15510 20 30 40 50 60 70 80 90 100 110 120 130 140(1)

CGCTAGAGAGGAGACGCATGTGTCAAAGTGTCCACTTTCAACCCCCTCACTCGCTTAAATATATTATTATTTTTAAATACTTCCTATATCCCATTTTTCACAAAGAGTGTGTTGCGAGATTAAAATATTTAGCCCACAATGAGCAAATATAAAAASmAn-gDNA (1)

-----------------------------------------------------------------------------------------------------------------------------------------------------------gSmMYB9 (1)

Consensus (1)

155 309160 170 180 190 200 210 220 230 240 250 260 270 280 290(155)

AACTCCCCCTCTCTCTCTCTCTCACATGCCAACATTTTCAAACCTCTCCTGTTCCATCTATATAAAGCCCCTCCCTCCTTTATCCTCTTCCACATCAACACAACCCCACCCCCTTTCTCTCTCTACATCTATACATCACCACCACTTTCTCTCTCSmAn-gDNA (155)

-----------------------------------------------------------------------------------------------------------------------------------------------------------gSmMYB9 (1)

Consensus (155)

310 464320 330 340 350 360 370 380 390 400 410 420 430 440 450(310)

CACACCATCATCAACTTTTCTTGTTGAGGGAGGATTCCTAGAGAGCGAAATTAACACGAGATGGATCATCACCAAGATGACACAAAATCATCAAGAAATTACAGCCAAAGTGAAGAAGATTTGTTGGAGCTGAGGAGAGGGCCATGGACAGTTGASmAn-gDNA (310)

------------------------------------------------------------ATGGATCATCACCAAGATGACACAAAATCAT---------ACAGCCAAAGTGAAGAAGATTTGTTGGAGCTGAGGAGAGGGCCATGGACAGTTGAgSmMYB9 (1)

ATGGATCATCACCAAGATGACACAAAATCAT ACAGCCAAAGTGAAGAAGATTTGTTGGAGCTGAGGAGAGGGCCATGGACAGTTGAConsensus (310)

465 619470 480 490 500 510 520 530 540 550 560 570 580 590 600(465)

TGAAGATTTTACTCTCATCAACTATATAGCCCACCATGGCGAAGGCCGTTGGAATTCTCTAGCTCGCTCTGCAGGTAATACTATTAATTCAAATTCTTCCTTTCCTTTCTAAATAAAAAAGTTTTTTTTTTTTTTTTTTTTTTTTTCATTTTAGTSmAn-gDNA (465)

TGAAGATTTTACTCTCATCAACTATATAGCCCACCATGGCGAAGGCCGTTGGAATTCTCTAGCTCGCTCTGCAGGTAATACTATTAATTCAAATTCTTCCTATCTTTTCTAAATAAAAAAGATTTTTTTTTTC-------TTTTTTCATTTTAGTgSmMYB9 (87)

TGAAGATTTTACTCTCATCAACTATATAGCCCACCATGGCGAAGGCCGTTGGAATTCTCTAGCTCGCTCTGCAGGTAATACTATTAATTCAAATTCTTCCT TC TTTCTAAATAAAAAAG TTTTTTTTTT TTTTTTCATTTTAGTConsensus (465)

620 774630 640 650 660 670 680 690 700 710 720 730 740 750 760(620)

TAGTTAGAGATAAGTGTTGATGATATATGCTCGATGTCCGTACAAGAAAAGAATTCGAGGCTAAATCCACTAAAAAGAGCATACAAAAGGTCCAAGAATTTACGTGTGAAGCTTTACCAATTAAAGCTGGTTATTATTTTCTTCTTTTCTTTGTTSmAn-gDNA (620)

TAGTTAGAGATAAGTGTTGATGATATATGCTCGATGTCCGTACAAGAAAAGAATTTGAGGCTAAATCCACTAAAAAAAGCATACAAAAGGTCCAAGAATTTACGTGTGAAACTTTACCAATTAAAGCTTGTTATTATTTTCTTCTTTTCTTTGTTgSmMYB9 (235)

TAGTTAGAGATAAGTGTTGATGATATATGCTCGATGTCCGTACAAGAAAAGAATT GAGGCTAAATCCACTAAAAA AGCATACAAAAGGTCCAAGAATTTACGTGTGAA CTTTACCAATTAAAGCT GTTATTATTTTCTTCTTTTCTTTGTTConsensus (620)

775 929780 790 800 810 820 830 840 850 860 870 880 890 900 910(775)

TCAATAATTTTTGGTGGTGAAGTAAACAAAATAAATTGATTGATTGATT----CAGGTTTGAAGAGAACCGGCAAGAGCTGCAGATTGAGGTGGCTAAACTACCTGCGGCCCGACGTCCGCCGCGGAAACATCACTCTCGAGGAGCAGCTGCTCASmAn-gDNA (775)

TCAATAATTTTTGGTGGCGAAGTAAACAAAATAAATTGATTGATTGATTGATTCAGGTTTGAAGAGAACCGGCAAGAGCTGCAGATTGAGGTGGCTAAACTACCTGCGGCCCGACGTCCGCCGCGGAAACATCACTCTCGAGGAGCAGCTGCTCAgSmMYB9 (390)

TCAATAATTTTTGGTGG GAAGTAAACAAAATAAATTGATTGATTGATT CAGGTTTGAAGAGAACCGGCAAGAGCTGCAGATTGAGGTGGCTAAACTACCTGCGGCCCGACGTCCGCCGCGGAAACATCACTCTCGAGGAGCAGCTGCTCAConsensus (775)

934 1088940 950 960 970 980 990 1000 1010 1020 1030 1040 1050 1060 1070(934)

CGAACTCCATTCTCGTTGGGGAAATAGGTACATACATATTAATTTTCCTAATTAATTAACTAATGTTATTATATATATGTAGGTCTGAAATTTAATTTCCAACTGATTGTATA-TATAAATTAATGTGTAGGTGGTCGAAAATAGCACAACATTTSmAn-gDNA (930)

CGAACTCCATTCTCGTTGGGGAAATAGGTGCATACATATTAATTTTCCTAATTAATTAACTAATGTTATTATATATATGTAGGTTTGAAATTTAATTTCCAACTGATTGTATAATATAAATTAATGTATAGGTGGTCGAAAATAGCACAACATTTgSmMYB9 (549)

CGAACTCCATTCTCGTTGGGGAAATAGGT CATACATATTAATTTTCCTAATTAATTAACTAATGTTATTATATATATGTAGGT TGAAATTTAATTTCCAACTGATTGTATA TATAAATTAATGT TAGGTGGTCGAAAATAGCACAACATTTConsensus (934)

1094 12481100 1110 1120 1130 1140 1150 1160 1170 1180 1190 1200 1210 1220 1230(1094)

GAAGGACGGACAACGAGATCAAGAACTACTGGCGCACGCGTGTGCAAAAGCATGCTAAGCAGCTGAAATGTGACGTGAACAGCAAGCAATTCAAGGACACCATGCGCTACCTATGGATGCCAAGATTGGTGGAGCGCATCCAAGCCGCCGCCGCCSmAn-gDNA (1089)

GAAGGACGGACAACGAGATCAAGAACTACTGGCGCACGCGTGTGCAAAAGCATGCTAAGCAGCTCAAATGTGACGTGAACAGCAAGCAATTCAAGGACACCATGCGCTACCTATGGATGCCCAGATTGGTGGAGCGCATCCAAGCCGCCGCCGCCgSmMYB9 (709)

GAAGGACGGACAACGAGATCAAGAACTACTGGCGCACGCGTGTGCAAAAGCATGCTAAGCAGCT AAATGTGACGTGAACAGCAAGCAATTCAAGGACACCATGCGCTACCTATGGATGCC AGATTGGTGGAGCGCATCCAAGCCGCCGCCGCCConsensus (1094)

1254 14081260 1270 1280 1290 1300 1310 1320 1330 1340 1350 1360 1370 1380 1390(1254)

CCCAAATCTCGACGTGGCCGTGGCCCTGCCGCGCCCCGAGAATTCGAGCGCGGCCGCGTCGTCGGAGTCCTTCGGGACGCCCGCGTCGGACCTGACGGATTGCTACAACAATCAGGATTGCTATGCGGCAAATAATAATCAGTTTAGCTATGGCGSmAn-gDNA (1249)

CCCGAATCTCGACGTGGCCGTGGCCCTGCCGCGCCCCGAGAATTCGAGCGCGGCCGCGTCGTCGGAGTCCTTCGGGACGCCCGCGTCGGACCTGACGGATTGCTACAACAATCAGGATTGCTATGCGGCAAATAATAATCAGTTTAGCTATGGCGgSmMYB9 (869)

CCC AATCTCGACGTGGCCGTGGCCCTGCCGCGCCCCGAGAATTCGAGCGCGGCCGCGTCGTCGGAGTCCTTCGGGACGCCCGCGTCGGACCTGACGGATTGCTACAACAATCAGGATTGCTATGCGGCAAATAATAATCAGTTTAGCTATGGCGConsensus (1254)

1409 15631420 1430 1440 1450 1460 1470 1480 1490 1500 1510 1520 1530 1540 1550(1409)

ATCAGTTGTCGCTAAGCAGCCCAAGTGGATACTTCAATCAAGGGCTGGATTTCGGCGGGCAGTGGGCGATGGACGGCGGAGATGCGTCGGACAGTTTGTGGAGCGTGGATGACGTGTGGTTCTTGCAGCAGCAGTTCAACACCAATGCTTGACTTSmAn-gDNA (1404)

ATCAATTGTCGCTAAGCAGCCCAAGTGGATACTTCAATCAAGGGCTGGATTTCGGCGGGCAGTGGGCGATGGACGGCGGAGATGCGTCGGACAGTTTGTGGAGCGTGGATGACGTGTGGTTCTTGCAGCAGCAGTTCAACACCAATGCTTGA---gSmMYB9 (1024)

ATCA TTGTCGCTAAGCAGCCCAAGTGGATACTTCAATCAAGGGCTGGATTTCGGCGGGCAGTGGGCGATGGACGGCGGAGATGCGTCGGACAGTTTGTGGAGCGTGGATGACGTGTGGTTCTTGCAGCAGCAGTTCAACACCAATGCTTGA Consensus (1409)

1570 17241580 1590 1600 1610 1620 1630 1640 1650 1660 1670 1680 1690 1700 1710(1570)

CATATATAGTTATTAGTTCATCAACATTTCATCACAAAGGATACATAAACCCTAGAAAAATAGGTTCTCTTCTTCATAGTATTTGTGGAGATCAGGGACAAGCTCATTCAACTTTTGATGATATATAGTACTAGTTTTTATGCTTGTAATTCTTTSmAn-gDNA (1565)

-----------------------------------------------------------------------------------------------------------------------------------------------------------gSmMYB9 (1176)

Consensus (1570)

1674 18281680 1690 1700 1710 1720 1730 1740 1750 1760 1770 1780 1790 1800 1810(1674)

CATTCAACTTTTGATGATATATAGTACTAGTTTTTATGCTTGTAATTCTTTGTTTCTTCTTTTTTTCTTTAAAAAAAAAAGTTTGTGGGGGATTTAATGAAGAGAAATTGTAACTTGATCATGCTAGCTACGTGAATTCAAATTCACTTCAAGCGSmAn-gDNA (1669)

-----------------------------------------------------------------------------------------------------------------------------------------------------------gSmMYB9 (1176)

Consensus (1674)

C

B

A

SmAn-gDNA

1823 bp

5'-UTR Intron 1 Intron 2 3'-UTR

Promoter Exon 1 Exon 2 Exon 3

225nt 169nt 288nt 130nt 103nt 493nt 271nt144nt

B1 90

SmMYB9b-ORF (1) ATGGATCATCACCAAGATGACACAAAATCATCAAGAAATTACAGCCAAAGTGAAGAAGATTTGTTGGAGCTGAGGAGAGGGCCATGGACASmMYB9-ORF (1) ATGGATCATCACCAAGATGACACAAAATCAT---------ACAGCCAAAGTGAAGAAGATTTGTTGGAGCTGAGGAGAGGGCCATGGACAConsensus (1) ATGGATCATCACCAAGATGACACAAAATCAT ACAGCCAAAGTGAAGAAGATTTGTTGGAGCTGAGGAGAGGGCCATGGACA

91 180SmMYB9b-ORF (91) GTTGATGAAGATTTTACTCTCATCAACTATATAGCCCACCATGGCGAAGGCCGTTGGAATTCTCTAGCTCGCTCTGCAGGTTTGAAGAGASmMYB9-ORF (82) GTTGATGAAGATTTTACTCTCATCAACTATATAGCCCACCATGGCGAAGGCCGTTGGAATTCTCTAGCTCGCTCTGCAGGTTTGAAGAGAConsensus (91) GTTGATGAAGATTTTACTCTCATCAACTATATAGCCCACCATGGCGAAGGCCGTTGGAATTCTCTAGCTCGCTCTGCAGGTTTGAAGAGA

181 270SmMYB9b-ORF (181) ACCGGCAAGAGCTGCAGATTGAGGTGGCTAAACTACCTGCGGCCCGACGTCCGCCGCGGAAACATCACTCTCGAGGAGCAGCTGCTCATTSmMYB9-ORF (172) ACCGGCAAGAGCTGCAGATTGAGGTGGCTAAACTACCTGCGGCCCGACGTCCGCCGCGGAAACATCACTCTCGAGGAGCAGCTGCTCATTConsensus (181) ACCGGCAAGAGCTGCAGATTGAGGTGGCTAAACTACCTGCGGCCCGACGTCCGCCGCGGAAACATCACTCTCGAGGAGCAGCTGCTCATT

271 360SmMYB9b-ORF (271) CTCGAACTCCATTCTCGTTGGGGAAATAGGTGGTCGAAAATAGCACAACATTTACCGGGAAGGACGGACAACGAGATCAAGAACTACTGGSmMYB9-ORF (262) CTCGAACTCCATTCTCGTTGGGGAAATAGGTGGTCGAAAATAGCACAACATTTACCGGGAAGGACGGACAACGAGATCAAGAACTACTGGConsensus (271) CTCGAACTCCATTCTCGTTGGGGAAATAGGTGGTCGAAAATAGCACAACATTTACCGGGAAGGACGGACAACGAGATCAAGAACTACTGG

361 450SmMYB9b-ORF (361) CGCACGCGTGTGCAAAAGCATGCTAAGCAGCTGAAATGTGACGTGAACAGCAAGCAATTCAAGGACACCATGCGCTACCTATGGATGCCASmMYB9-ORF (352) CGCACGCGTGTGCAAAAGCATGCTAAGCAGCTCAAATGTGACGTGAACAGCAAGCAATTCAAGGACACCATGCGCTACCTATGGATGCCCConsensus (361) CGCACGCGTGTGCAAAAGCATGCTAAGCAGCT AAATGTGACGTGAACAGCAAGCAATTCAAGGACACCATGCGCTACCTATGGATGCC

451 540SmMYB9b-ORF (451) AGATTGGTGGAGCGCATCCAAGCCGCCGCCGCCGCCGCCCCAAATCTCGACGTGGCCGTGGCCCTGCCGCGCCCCGAGAATTCGAGCGCGSmMYB9-ORF (442) AGATTGGTGGAGCGCATCCAAGCCGCCGCCGCCGCCGCCCCGAATCTCGACGTGGCCGTGGCCCTGCCGCGCCCCGAGAATTCGAGCGCGConsensus (451) AGATTGGTGGAGCGCATCCAAGCCGCCGCCGCCGCCGCCCC AATCTCGACGTGGCCGTGGCCCTGCCGCGCCCCGAGAATTCGAGCGCG

541 630SmMYB9b-ORF (541) GCCGCGTCGTCGGAGTCCTTCGGGACGCCCGCGTCGGACCTGACGGATTGCTACAACAATCAGGATTGCTATGCGGCAAATAATAATCAGSmMYB9-ORF (532) GCCGCGTCGTCGGAGTCCTTCGGGACGCCCGCGTCGGACCTGACGGATTGCTACAACAATCAGGATTGCTATGCGGCAAATAATAATCAGConsensus (541) GCCGCGTCGTCGGAGTCCTTCGGGACGCCCGCGTCGGACCTGACGGATTGCTACAACAATCAGGATTGCTATGCGGCAAATAATAATCAG

631 720SmMYB9b-ORF (631) TTTAGCTATGGCGATCAGTTGTCGCTAAGCAGCCCAAGTGGATACTTCAATCAAGGGCTGGATTTCGGCGGGCAGTGGGCGATGGACGGCSmMYB9-ORF (622) TTTAGCTATGGCGATCAATTGTCGCTAAGCAGCCCAAGTGGATACTTCAATCAAGGGCTGGATTTCGGCGGGCAGTGGGCGATGGACGGCConsensus (631) TTTAGCTATGGCGATCA TTGTCGCTAAGCAGCCCAAGTGGATACTTCAATCAAGGGCTGGATTTCGGCGGGCAGTGGGCGATGGACGGC

721 795SmMYB9b-ORF (721) GGAGATGCGTCGGACAGTTTGTGGAGCGTGGATGACGTGTGGTTCTTGCAGCAGCAGTTCAACACCAATGCTTGASmMYB9-ORF (712) GGAGATGCGTCGGACAGTTTGTGGAGCGTGGATGACGTGTGGTTCTTGCAGCAGCAGTTCAACACCAATGCTTGAConsensus (721) GGAGATGCGTCGGACAGTTTGTGGAGCGTGGATGACGTGTGGTTCTTGCAGCAGCAGTTCAACACCAATGCTTGA

C1 90

SmMYB9b (1) MDHHQDDUKSSRNYSQSEEDLLELRRGPWUVDEDFULINYIAHHGEGRWNSLARSAGLKRUGKSCRLRWLNYLRPDVRRGNIULEEQLLISmMYB9 (1) MDHHQDDTKS---YSQSEEDLLELRRGPWTVDEDFTLINYIAHHGEGRWNSLARSAGLKRTGKSCRLRWLNYLRPDVRRGNITLEEQLLI

Consensus (1) MDHHQDDTKS YSQSEEDLLELRRGPWTVDEDFTLINYIAHHGEGRWNSLARSAGLKRTGKSCRLRWLNYLRPDVRRGNITLEEQLLI91 180

SmMYB9b (91) LELHSRWGNRWSKIAQHLPGRUDNEIKNYWRURVQKHAKQLKCDVNSKQFKDUMRYLWMPRLVERIQAAAAAAPNLDVAVALPRPENSSASmMYB9 (88) LELHSRWGNRWSKIAQHLPGRTDNEIKNYWRTRVQKHAKQLKCDVNSKQFKDTMRYLWMPRLVERIQAAAAAAPNLDVAVALPRPENSSA

Consensus (91) LELHSRWGNRWSKIAQHLPGRTDNEIKNYWRTRVQKHAKQLKCDVNSKQFKDTMRYLWMPRLVERIQAAAAAAPNLDVAVALPRPENSSA181 264

SmMYB9b (181) AASSESFGUPASDLUDCYNNQDCYAANNNQFSYGDQLSLSSPSGYFNQGLDFGGQWAMDGGDASDSLWSVDDVWFLQQQFNUNASmMYB9 (178) AASSESFGTPASDLTDCYNNQDCYAANNNQFSYGDQLSLSSPSGYFNQGLDFGGQWAMDGGDASDSLWSVDDVWFLQQQFNTNA

Consensus (181) AASSESFGTPASDLTDCYNNQDCYAANNNQFSYGDQLSLSSPSGYFNQGLDFGGQWAMDGGDASDSLWSVDDVWFLQQQFNTNA

11

Fig. S3 PCR screening for SmMYB9b over expressing hairy roots of S. miltiorrhiza.

Lane 1: actin and rolC with genomic DNA of danshen plants as template. Lane 2: SmMYB9b

with genomic DNA of danshen plants as template. Lane 3: actin and rolC with genomic DNA

of control hairy roots as template. Lane 4: SmMYB9b with genomic DNA of control hairy

roots as template. Lane 5: actin and rolC with genomic DNA of SmMYB9b-OX hairy roots as

template. Lane 6: SmMYB9b with genomic DNA of transgenic hairy roots as template. Lanes

1’ to 35’: PCR products of actin and rolC (left) and SmMYB9b (right) in individual

SmMYB9b-OX danshen hairy root lines.

1’ 2’ 3’ 4’ 5’ 6’ 7’ 9’ 10’

11’ 12’ 13’ 14’ 16’ 21’ 32’ 33’ 35’

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Fig. S4 Relative expression level of SmMYB9b in individual SmMYB9b-OX hairy root lines

Wild type danshen hairy roots were used as the reference sample. All data are the means of

three independent replicates with error bars indicating the SD. SmMYB9b-OX hairy root lines

were displayed in a descending order according to the relative expression level of SmMYB9b.

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Fig. S5 Morphology of wild type danshen hairy roots after treatment with ABA and GA

One gram of fresh wild hairy roots were inoculated into MSOH media supplemented with

100µM of ABA (C, D), 100µM of GA (E, F), or 100µM of MeJA (G, H) and cultured for three

weeks before collected. Wild type hairy roots cultured in MSOH media supplemented with

the same volume of 50% DMSO was used as mock samples (A, B).